Method and apparatus for rapid measurement of HbA1c

ABSTRACT

Described is a method and apparatus for determining a diabetic patient&#39;s compliance with their insulin dosing regime. The method and apparatus involves taking a blood sample from a patient by a routine finger prick and placing it in a special sample tab which is placed in a spectrophotometer sample housing. The spectrophotometer measures Hb and HbA 1c  concentrations and allows for calculating a ratio of HbA 1c  to Hb which is indicative of the degree of patient compliance.

This application claims priority to International Application No.PCT/CA00/00549 filed May 11, 2000, which in turn claims priority to U.S.Provisional Application No. 60/133,876, filed May 12, 1999.

FIELD OF INVENTION

This invention relates to a sample tab and sample housing for performingrapid spectrophotometric measurement of Hemoglobin A_(1c) (HbA_(1c)) inwhole blood, without the use of any reagent.

BACKGROUND OF INVENTION

Diabetes mellitus is due to absolute or relative insulin deficiency. Themost common forms of diabetes are Type 1 or Insulin-dependent diabetes,and Type 2 or Non-insulin-dependent diabetes. What all forms of diabeteshave in common is elevation in blood glucose or hyperglycemia. There areabout 16 million diabetics in the US, with about 10-15% being Type 1 andthe rest being Type 2. Type 1 diabetes is caused by an absolute insulindeficiency, and usually occurs before the age of 30, although it canoccur at any age. Consequently, it was also referred to as juvenilediabetes. It is not associated with obesity and is commonly complicatedby ketoacidosis. Ketoacidosis is an acute complication of diabetes, andmay present as a medical emergency because of dehydration and acidosis(low blood pH). Type 2 diabetes usually develops after the age of 30 andis not associated with total loss of the ability to secrete insulin.Consequently, it was referred to as maturity-onset diabetes. Plasmainsulin levels are often normal or elevated. Al most all the patientsare obese, and their glucose tolerance may be restored to normal if theyloose weight. They have a reduced number of insulin receptors, and thenumber of these receptors can increase with weight loss. Due to thepresence of circulating insulin, ketoacidosis is a rare complication.

The late complications of all forms of diabetes are kidney failure(nephropathy), blindness (due to retinopathy), sensory deficits (due toneuropathy). Recent long-term clinical evaluations report that failureof a patient to maintain glucose levels as close to normal as possiblecan contribute to these significant complications of diabetes. Toadequately control the glucose levels in their blood, diabetic patientsmust inject themselves with insulin once or twice daily, and mustmonitor their blood glucose levels between 1 and 4 times daily. The mostcommon method used by diabetic patients for monitoring blood glucose, isto acquire a small sample of blood by sticking the finger with a lancet,and squeezing a droplet of blood onto a paper strip which is then placedon a detection device. The glucose results assist the patients inplaning meals and physical activities, and also assist the doctors inoptimizing the patients' insulin dosage. Unfortunately, many diabeticpatients are not compliant in measuring their blood glucose regularly,and regulating their diet and physical activities, but yet their glucoselevels may be at acceptable levels during their visit to the doctor'soffice. To get around this problem in detecting non-compliance, doctorsmonitor their patients' HbA_(1c) levels every 2 to 4 months.

HbA_(1c) is one specific type of glycated Hb, constituting approx. 80%of all glycated Hb and is formed by the spontaneous reaction of glucosewith the N-terminal amino group of the Hb A beta chain. The HbA_(1c) andthe glycated Hb values have a high degree of correlation, and either maybe used in the management of diabetes. As a matter of fact, some invitro diagnostic systems measure glycated Hb but report HbA_(1c)results. Formation of HbA_(1c) irreversible, and the blood level dependson both the life span of the red blood cells (average 120 days) and theblood glucose concentration. Therefore HbA_(1c) represents thetime-averaged blood glucose values over the preceding 4 to 6 weeks, andis not subject to the wide fluctuations observed in blood glucosevalues. Studies have shown that quality of life improves with decreasinglevels of HBA_(1c), and measurements every 2 to 4 months arerecommended.

The gold standard for measuring HbA1c uses high performance liquidchromatography (HPLC). Other methods use affinity chromatography,ion-exchange chromatography and immunoinhibition turbidimetrictechniques. In all the available methods, the first step is theproduction of a hemolysate by lysing the red blood cells with a specialreagent. Since no near-patient testing for HbA_(1c) is currentlyavailable, diabetic patients have to visit their doctor a second time todiscuss their HbA_(1c) results. The inconvenience to patients and theextra cost for a follow-up visit to the doctor, prompted manufacturersto develop a kit, which enables the patient to place their blood on aspecially-treated test strip, which is then sent to a laboratory in aprepaid mailer. Within 1 to 2 weeks, both patients and their doctorsreceive the HbA_(1c) results. By mailing in a blood sample ahead oftime, the follow-up visit to the doctor can be eliminated. A rapidmethod for performing the HbA_(1c) test in the doctors office is stillpreferred.

SUMMARY OF THE INVENTION

It is desirable to provide an apparatus and a method whereby a doctorcan test his/her patent's HbA_(1c) within minutes. It is preferred thatthe sample requirement is a drop of blood drawn by finger prick, in amanner comparable to near-patient glucose testing. The advantages of thepresent invention are the rapid turn-around time during a patient'svisit with his/her doctor, and the decreased costs due to absence ofreagents.

In its broad aspect the present invention provides an apparatus fordetermining the concentration of HbA_(1c) and Hb in a blood specimenwhere the apparatus comprises: a sample tab; a sample housing forreceiving a sample; and a radiation source and radiation detector,operatively coupled with a means for providing a determination ofglucose concentration in the blood sample based on the absorbedradiation.

According to one embodiment of the present invention, the sample housingcomprises a block with a slit for inserting the sample tab, and morepreferably, the sample tab consists of a slide or base plate with adepression or well in the base plate for containing the sample and acoverslip which closes when the tab is inserted in the housing,preferably, the cover closes automatically when inserted in the samplehousing.

In a preferred embodiment of the present invention, the sample wellcontains two grooves and an overflow ring for collecting excess blood asit is squeezed out by the closing coverslip. Preferably, the coverslipis attached to the tab so that the blood proximate the coverslip hingemakes contact with the coverslip first; as the coverslip closes, excessblood is squeezed out through the two grooves and into the overflowring.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 is a perspective view of a system incorporating an apparatus ofthe present invention for measuring Hemoglobin A1c;

FIG. 2 is a perspective view illustrating the sample tab of theapparatus of FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of determining a diabeticpatient's compliance with their insulin dosing regime comprisingquantifying the amount of HbA_(1c) and Hb contained in a blood specimentaken from the patient, without further treatment of the specimen, usinga spectrophotometer, and comparing the concentration of HbA_(1c) and Hb,where an elevated ratio of HbA_(1c) reflects a lack of patientcompliance.

According to a preferred embodiment of the method of this invention, themethod of quantification comprises the steps of:

(i) generating a calibration algorithm for each of the HbA_(1c) and Hb;

(ii) measuring with the spectrophotometer, absorbance of radiation byeach of the HbA_(1c) and Hb in the specimen; and

(iii) incorporating the absorbances measured in step (ii) in thealgorithms respectively and calculating the concentration of theHbA_(1c) and Hb in the specimen.

More preferably, according to the method of the present invention,quantification includes calculation of the first derivatives of at leasttwo portions of a spectrum generated from a scan for each of HbA_(1c)and Hb which are used to calculate each of the HbA_(1c) and Hbconcentrations.

According to another aspect of the present invention, the methods can beused with reflectance instead of absorbance.

According to a preferred aspect of the present invention, the method iscarried out with a blood specimen being placed into a sample tabcomprising a well in which the specimen resides and a cover which closesover the well.

Furthermore, the method provides for the situation where the sample tabwell allows for overflow of excess specimen from the well whenever thecover is closed over the well.

Turning now to the sample housing and sample tab, as may be seen in FIG.1 the radiation from the spectrophotometer is delivered to the sample inhe sample tab through a source or incident optical fibre (60) while thesample rests in a sample tab holder (70) within a sample housing (80).The radiation passing through the sample tab and specimen is received bya receiving optical fiber (90), and processed further to determineconcentrations of Hb and HbA_(1c).

Spectrophotometer

A spectrophotometer of the present invention is one with appropriatefilters, a grating and a linear photodiode array (PDA) detector; a meansfor optically connecting the radiation source with the detector along asample path through the housing and along a reference path whichby-passes the sample; a means for selectively passing a beam from thesample path and from the reference path to the detector; a means forselecting an appropriate integration time required for adequate detectorresponse; and a means for correlating a detector response, from thesample path relative to a detector response from the reference path, toa quantity of HbA_(1c) or Hb, as appropriate, in said sample.

The apparatus further comprises a quartz-tungsten-halogen bulb capableof emitting a near infrared light beam having wavelengths from 600 nm to1100 nm and a single optical fiber bundle which randomly samples lightfrom the quartz-tungsten-halogen bulb. The single fiber bundlebifurcates into a sample path beam for travel along a sample path and areference path beam for travel along a reference path. The bifurcatedoptical fiber consists of multiple fibers which focus random sampling oflight from the lamp, into single fibers of 0.4 millimeter diameter forboth the sample and reference beams. This apparatus further comprisestwo shutters, installed in the lamp assembly, for selectively blockingthe sample path light beam which travels along the sample path through asample enclosed in a housing and the reference path light beam whichtravels along the reference path. The two light paths are collected intotwo fibers which converge into a single fiber which is focused onto thedetector; the bifurcated collection optical fiber consists of multiplefibers. This apparatus further comprises a grating for dispersing thecombined beam into component wavelengths which are passed onto thedetector. The detector of this apparatus is a silicon PDA comprised of aplurality of pixels wherein each of the pixels is set to measure one ofa plurality of predetermined light frequencies. Based on the measurementof the frequencies, the detector generates a plurality of signalswherein each of the signals is responsive to an amount of radiationreceived by each of the pixels. This apparatus further comprises ananalog-to-digital converter to generate digital information from theplurality of signals and a microprocessor, which is connected to theconverter, to correlate the digital information to a quantity of a knownsubstance in the sample.

Alternatively, an InGaAs (Indium-Gallium-Arsenide) PDA which covers thewavelength range of 800 nm to 1700 nm or 1200 nm to 2600 nm can be used,or any commercially available scanning near infrared spectrophotometerswhich covers the range of 700 nm to 2500 nm.

In another aspect of the invention, a light-tight sample housing is notrequired. The only shutters in the apparatus are the two located in thelamp assembly, and are used for sequentially directing the light throughthe sample or reference pathway. Since there is no shutter between thesample housing and the sensor, any room light leakage into the samplehousing will affect the sample light and sample dark scans equally whenperformed at the same integration tie, and also the reference light andreference dark scans when performed at the same integration time usedfor the reference measurements. Therefore, room light impinging on thedetector can be effectively subtracted without affecting the performanceof the apparatus, provided that the ambient light does not change duringthe few seconds measurement time. The room light leakage along sides ofthe tab, can be managed by measuring the dark current, i.e., detectorresponse when detector is not exposed to the instrument light, for boththe sample and reference measurements.

The PDA integrates the optical radiation over a specified time andconverts the optical signal to a time multiplexed analog electronicsignal called a scan where absorbance is calculated as:

 Absorbance_(i)=log{(Reference Light_(i)−Reference Dark_(i))/(SampleLight_(i)−Sample Dark_(i)}+log() ITS/ITR)

where

Absorbance_(i)=Absorbance pixel i

Reference Light_(i)=Reference pixel i readings, with reference path openand sample path closed by a shutter;

Reference Dark_(i)=Reference pixel i readings, with reference and samplepaths closed by shutters;

Sample Light_(i)=Sample pixel i readings, with sample path open andreference path closed by a shutter;

Sample Dark_(i)=Sample pixel i readings, with sample and reference pathsclosed by shutters;

ITS=Integration time for sample measurement;

ITR=Integration time for reference measurement; and

i=the particular pixel (wavelength) in the PDA.

The electronic signal is proportional to the time that the detectorintegrates the optical signal. The electronic signal is amplified byanalog electronic amplifiers and converted to a digital signal by ananalog-to-digital converter or ADC. The digital information from theconverter is interpreted for data analysis by a microprocessor which isin turn connected via an RS232 connector to a computer. The results ofthe data analysis can be displayed on the computer, or on a printerconnected.

The integration time for the sample beam is low for a sample with lowhematocrit, since there is less scattered light and therefore more lightis transmitted to the detector. When the light is sufficiently scatteredby, for example a high hematocrit, the spectrophotometer willautomatically switch to a higher integration time. The higherintegration time chosen will be within a pre-selected range, such thatthe detector's response is optimal. This feature will allow all samples,from the lowest to the highest hematocrit, to be efficiently testedwithout exceeding the linear response range of the detector.

Sample Tab

According to another aspect of the present invention, there is provideda sample tab for use in monitoring a diabetic patient's compliance withtheir insulin dosing regime by spectrophotometry of a blood specimenfrom the patient, the tab comprising:

a base plate having a top and bottom surface,

a well in the top surface,

the upper portion of the well being defined by a closed wall extendingabove the top surface of the plate,

at least one notch in the wall to allow drainage of excess blood, and acover plate,

the cover plate and base plate being translucent where the sampleresides in the well to allow radiation to be transmitted through thecover plate, blood specimen and the base plate.

According to a further embodiment, the wall of the well is surrounded bya second closed wall to retain excess blood drained from the well,preferably the cover is attached to the base plate.

Referring now to FIG. 2, in a preferred embodiment of a sample tab ofthe present invention (5), the sample cavity or “well” (10) is 2millimeter deep and 4 millimeters diameter, i.e., of sufficient size toallow a drop of blood fill the sample cavity, with some excess. Smalloverflow grooves (20) allow excess blood to flow out of the well. Anoverflow ring (30) retains any overflow blood from running off the tab.The cover (40) is in a preferred embodiment attached to the tab by ahinge (50). The entire tab may be conveniently manufactured from anysuitable plastic material. In the prototype, black plastic washers wit2-centimeter internal diameter and 2-millimeter thickness were glued tomicroscope slides, and microscope coverslips were used to cover thesamples. Also, a microscope was used as the sample housing after thefollowing modification: the input fiber was sent through the condenserposition, and the output fiber was sent through the objective lens; boththe condenser and objective were replaced with machined fixtures whichhoused the ends of the fibres. A microscope stage was used for holdingand positioning the slide. For the prototype, 350 μL of whole blood wasused; for the preferred embodiment, 25 μL would be sufficient. However,the volume of the sample cavity should not be a restriction for thepresent invention.

The tabs and coverslips can be made of glass as used in microscopy, butplastic is preferred. The plastic can be transparent or translucent. Apreferred plastic is polypropylene, which is translucent.

By virtue of the orientation of the sample housing (80), the projectionof light is in the vertical direction. An advantage of this is that thered blood cells will remain m the light path, even as they falldownwards under the effect of gravity. It will be obvious to thoseskilled in the art, that a flow-through cuvette lie those inCO-oximeters can also be used.

Calibration

As with any quantitative method, calibration of the spectrophotometer isrequired. However the methods for NIR calibration is much more complexthan most which can be calibrated with a minimum of a single standardmaterial of known concentration. In respect of NIR calibration, samplesmust contain all spectral variability expected during the analysis of anunknown sample; the sample must contain an even distribution of theanalyte of interest, and the concentrations of total Hb should notcorrelate significantly with HbA_(1c). The development of the algorithmuses PLS (Partial Least Squares) analysis of the full spectrum. A samplesize of several hundred samples is necessary to characterize all thesample variability, particularly due to the various Hb species.

The three parameters measured are grams/liter total hemoglobin (Hb),grams/liter HbA_(1c), and %HbA_(1c). Because % HbA_(1c) is a ratio ofHbA_(1c) to total Hb multiplied by 100, % HbA_(1c) is not affected byartifactual dilution caused by institial fluids squeezed out with theblood, when the finger is “milked” for the blood. Similarly, theimprecision in the manufacture of the tabs, in particular with respectto path length, will not affect the % HbA_(1c).

While the present invention has been described with reference to whatare presently considered to be preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalents included within the spirit and scope ofthe appended claims

We claim:
 1. A method of determining a diabetic patient's compliancewith their insulin dosing regime comprising: (i) obtaining an untreatedblood specimen from said patient; (ii) measuring with aspectrophotometer, absorbance of radiation by each of HbA_(1c) and totalHb in said specimen, the radiation being directed into said specimen ina direction that is substantially parallel to the direction in which redblood cells in said specimen fall under the effect of gravity; (iii)using a calibration algorithm for each of said HbA_(1c) and total Hb tocalculate a concentration of each of said HbA_(1c) and total Hb in saidspecimen from said absorbances measured in step (ii); and (iv)determining a ratio between the concentrations of HbA_(1c) and total Hb,wherein an elevated concentration ratio of HbA_(1c) to total Hb reflectsa lack of patient compliance.
 2. The method of claim 1, wherein saidstep of using (step iii), includes a step for calculating a firstderivative of at least two portions of a spectrum generated from anabsorbance scan for each of HbA_(1c) and total Hb.
 3. The method ofclaim 1, wherein said spectrophotometer measures reflectance instead ofabsorbance.
 4. The method of claim 1, wherein prior to said step ofmeasuring (step ii), said specimen is placed into a sample tab, and thesample tab is inserted into said spectrophotometer, wherein said sampletab comprises a well in which the specimen resides and a cover whichcloses over the well, and wherein said sample tab is transparent ortranslucent.
 5. A method of determining a diabetic patient's compliancewith their insulin dosing regime comprising: (i) generating acalibration algorithm for each of HbA_(1c) and total Hb; (ii) obtainingan untreated blood specimen from said patient; (iii) measuring with aspectrophotometer, absorbance of radiation by each of HbA_(1c) and totalHb in said specimen, the radiation being directed into said specimen ina direction that is substantially parallel to the direction in which redblood cells in said specimen fall under the effect of gravity; (iv)using said calibration algorithms generated in step (i) to calculate aconcentration of each of said HbA_(1c) and total Hb in said specimenfrom said absorbances measured in step (iii); and (v) determining aratio between the concentrations of HbA_(1c) and total Hb, wherein anelevated concentration ratio of HbA_(1c) to total Hb reflects a lack ofpatient compliance. 6.The method of claim 5, wherein said step of using(step iv), includes a step for calculating a first derivative of atleast two portions of a spectrum generated from an absorbance scan foreach of HbA_(1c) and total Hb.
 7. The method of claim 5, wherein saidspectrophotometer measures reflectance instead of absorbance.
 8. Themethod of claim 5, wherein prior to said step of measuring (step iii),said specimen is placed into a sample tab, and the sample tab isinserted into said spectrophotometer, wherein said sample tab comprisesa well in which the specimen resides and a cover which closes over thewell, and wherein said sample tab is transparent or translucent.
 9. Amethod of assessing the control of diabetes in a patient comprising: (i)obtaining an untreated blood specimen from said patient; (ii) measuringwith a spectrophotometer, absorbance of radiation by each of HbA_(1c)and total Hb in said specimen, the radiation being directed into saidspecimen in a direction that is substantially parallel to the directionin which red blood cells in said specimen fall under the effect ofgravity; (iii) using a calibration algorithm for each of said HbA_(1c)and total Hb to calculate a concentration of each of said HbA_(1c) andtotal Hb in said specimen from said absorbances measured in step (ii);and (iv) determining a ratio between the concentrations of HbA_(1c) andtotal Hb, wherein an elevated concentration ratio of HbA_(1c) to totalHb reflects a lack of control of diabetes.
 10. The method of claim 9,wherein said step of using (step iii), includes a step for calculating afirst derivative of at least two portions of a spectrum generated froman absorbance scan for each of HbA_(1c) and total Hb.
 11. The method ofclaim 9, wherein said spectrophotometer measures reflectance instead ofabsorbance.
 12. The method of claim 9, wherein said diabetes is Type 1or Type 2 diabetes.
 13. The method of claim 9, wherein prior to saidstep of measuring (step ii), said specimen is placed into a sample tab,and the sample tab is inserted into said spectrophotometer, wherein saidsample tab comprises a well in which the specimen resides and a coverwhich closes over the well, and wherein said sample tab is transparentor translucent.
 14. A method of assessing the control of diabetes in apatient comprising: (i) obtaining an untreated blood specimen from saidpatient; (ii) measuring with a spectrophotometer, absorbance ofradiation by each of HbA_(1c) and total Hb in said specimen, theradiation being directed into said specimen in a direction that issubstantially parallel to the direction in which red blood cells in saidspecimen fall under the effect of gravity; and (iii) using a calibrationalgorithm to calculate a concentration ratio of said HbA_(1c) to totalHb in said specimen from said absorbances measured in step (ii), whereinan elevated ratio of HbA_(1c) to total Hb reflects a lack of control ofdiabetes.
 15. The method of claim 14, wherein said step of using (stepiii), includes a step for calculating a first derivative of at least twoportions of a spectrum generated from an absorbance scan for each ofHbA_(1c) and total Hb.
 16. The method of claim 14, wherein saidspectrophotometer measures reflectance instead of absorbance.
 17. Themethod of claim 14, wherein said diabetes is Type 1 or Type 2 diabetes.18. The method of claim 14, wherein prior to said step of measuring(step ii), said specimen is placed into a sample tab, and the sample tabis inserted into said spectrophotometer, wherein said sample tabcomprises a well in which the specimen resides and a cover which closesover the well, and wherein said sample tab is transparent ortranslucent.
 19. A method of assessing the control of diabetes in apatient comprising: (i) generating a calibration algorithm for each ofHbA_(1c) and total Hb; (ii) obtaining an untreated blood specimen fromsaid patient; (iii) measuring with a spectrophotometer, absorbance ofradiation by each of HbA_(1c) and total Hb in said specimen, theradiation being directed into said specimen in a direction that issubstantially parallel to the direction in which red blood cells in saidspecimen fall under the effect of gravity; (iv) using said calibrationalgorithms generated in step (i) to calculate a concentration of each ofsaid HbA_(1c) and total Hb in said specimen from said absorbancesmeasured in step (iii); and (v) determining a ratio between theconcentrations of HbA_(1c) and total Hb, wherein an elevatedconcentration ratio of HbA_(1c) to total Hb reflects a lack of controlof diabetes.
 20. The method of claim 19, wherein said step of using(step iv), includes a step for calculating a first derivative of atleast two portions of a spectrum generated from an absorbance scan foreach of HbA_(1c) and total Hb.
 21. The method of claim 19, wherein saidspectrophotometer measures reflectance instead of absorbance.
 22. Themethod of claim 19, wherein said diabetes is Type 1 or Type 2 diabetes.23. The method of claim 19, wherein prior to said step of measuring(step iii), said specimen is placed into a sample tab, and the sampletab is inserted into said spectrophotometer, wherein said sample tabcomprises a well in which the specimen resides and a cover which closesover the well, and wherein said sample tab is transparent ortranslucent.
 24. A method of assessing the control of diabetes in apatient comprising: (i) generating a calibration algorithm for both ofHbA_(1c) and total Hb; (ii) obtaining an untreated blood specimen fromsaid patient; (iii) measuring with a spectrophotometer, absorbance ofradiation by each of HbA_(1c) and total Hb in said specimen, theradiation being directed into said specimen in a direction that issubstantially parallel to the direction in which red blood cells in saidspecimen fall under the effect of gravity; and (iv) using saidcalibration algorithms to calculate a concentration ratio of saidHbA_(1c) to total Hb in said specimen from said absorbances measured instep (iii), wherein an elevated concentration ratio of HbA_(1c) to totalHb reflects a lack of control of diabetes.
 25. The method of claim 24,wherein said step of using (step iv), includes a step for calculating afirst derivative of at least two portions of a spectrum generated froman absorbance scan for each of HbA_(1c) and total Hb.
 26. The method ofclaim 24, wherein said spectrophotometer measures reflectance instead ofabsorbance.
 27. The method of claim 24, wherein said diabetes is Type 1or Type 2 diabetes.
 28. The method of claim 24, wherein prior to saidstep of measuring (step iii), said specimen is placed into a sample tab,and the sample tab is inserted into said spectrophotometer, wherein saidsample tab comprises a well in which the specimen resides and a coverwhich closes over the well, and wherein said sample tab is transparentor translucent.